Scanning infrared measurement system

ABSTRACT

An analyzer of a component in a sample fluid includes an optical source and an optical detector defining a beam path of a beam, wherein the optical source emits the beam and the optical detector measures the beam after partial absorption by the sample fluid, a fluid flow cell disposed on the beam path defining an interrogation region in the a fluid flow cell in which the optical beam interacts with the sample fluid and a reference fluid; and wherein the sample fluid and the reference fluid are in laminar flow, and a scanning system that scans the beam relative to the laminar flow within the fluid flow cell, wherein the scanning system scans the beam relative to both the sample fluid and the reference fluid.

CROSS REFERENCE

This application is a continuation of U.S. patent application Ser. No.16/203,901, filed Nov. 29, 2018 and entitled, “Scanning InfraredMeasurement System”, which in turn is a continuation of U.S. patentapplication Ser. No. 15/048,705, filed Feb. 19, 2016, which in turnclaims benefit of U.S. provisional patent application No. 62/118,005,filed Feb. 19, 2015. The '901, '705, and '005 applications are herebyincorporated by reference herein in their entireties. U.S. patentapplication Ser. No. 13/894,831, filed May 15, 2013 and entitled,“Cytometry System with Interferometric Measurement”, is alsoincorporated by reference in its entirety into this application.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

Not applicable to this application.

BACKGROUND OF THE INVENTION 1. Field of the Invention

The present invention relates generally to infrared spectroscopy andmore specifically a system by which infrared lasers, including QCLs, maybe used to measure liquid samples, and provides significant advantagesin terms of signal-to-noise ratio in measuring chemical composition ofthese liquids; as well as resulting in a system that is very stable inthe face of laser or other optical train changes.

2. Any discussion of the prior art throughout the specification shouldin no way be considered an admission that such prior art is widely knownor forms part of common general knowledge in the field.

BACKGROUND

Infrared spectroscopy is a valuable, well-known tool for chemicalcharacterization of gaseous, liquid and solid substances becausecompounds have distinct absorption “fingerprints” in the mid-infraredregion, with absorption bands corresponding to vibrational energies ofmolecular bonds.

In theory, infrared spectroscopy should be a very valuable tool foranalyzing liquid samples for applications including, but not limited to:medical liquid analysis (blood, urine, saliva, etc.) for diagnostics orsubstance detection; industrial or food/beverage process control;pollutant detection; etc.

A major barrier to broader application of infrared spectroscopy toliquid samples has been the high inherent absorption of many liquids inthe infrared. For example, water has strong infrared absorption, makinganalysis of aqueous solutions difficult. A number of tools have beendeveloped to circumvent this issue, for example: the use of attenuatedtotal reflection (ATR) prisms and other surface-grazing opticaltechniques; drying of samples before analysis; and the use ofliquid-liquid extraction processes to transfer solutes from one liquidto another, more infrared-transparent liquid. Each of these introducespotential complexities and inaccuracies into measurements of liquids.

New and improved light sources in the infrared, including quantumcascade lasers (QCLs) offer significantly higher power at specificwavelengths of interest than traditional “glo-bar” (incandescentbroadband thermal emitting) sources. This higher power potentiallyenables absorption measurements in thicker liquid samples, whilemaintaining sufficient power throughput to allow reasonablesignal-to-noise for measurement of chemical concentrations in thesample. Measurements can then be performed with one or more wavelengths,with one or more “signal” wavelengths at absorption peaks of interest,and possibly wavelengths designed to provide reference or baselinelevels (off-peak). Multiple wavelengths may be achieved using multiplelasers, or through the use of wavelength-tunable sources.

For detection of low concentrations of compounds in liquids, or subtlechanges in chemical makeup, the incremental infrared absorptioncorresponding to concentrations of interest may be extremely small.Therefore even with higher power transmission, there remains the problemof detecting small absorption signals against a high background.

A solution employed to measure low concentrations in spectroscopy is theuse of reference wavelengths. For example, sample transmission at thewavelength corresponding to an absorption peak of a substance ofinterest is measured, together with the transmission at two nearbywavelengths, one higher and one lower. A “baseline” is then computedusing the reference wavelength transmissions, and the transmission atthe “peak” wavelength is divided by this baseline. This type of baselineadjustment can compensate for factors such as sample thickness, broadabsorption by other compounds, and detector responsivity changes. In thecase of broadband infrared sources, it also compensates—over a limitedwavelength range—for changes in source output. For example, suchreferencing will drastically reduce effects from changes in temperatureof a conventional blackbody thermal source. Indeed, this allowstraditional Fourier-Transform Infrared (FTIR) instruments equipped withglo-bar sources—or even using broadband radiation from synchrotronsources—to produce spectral data that may be locally baselined (inwavelength) to accurately determine chemical content.

Such baselining techniques, however, may be significantly less effectivewith infrared laser sources such as those that could deliver higherpower to penetrate thicker liquid samples. Laser sources are inherentlynarrowband, resonant devices, rather than broadband emitters. Theiroutput—power, wavelength, bandwidth, polarization and spatial beamproperties—can be highly sensitive to device and operating conditionsincluding current, temperature, aging, and feedback (from reflections).Moreover, any changes in these conditions may cause highly discontinuouschanges in output. Moreover, these changes will not be consistent fromone laser to another, or even from one wavelength to another in the caseof a broadband or tunable laser.

As a result, changes between illumination at the “peak” (absorption, ofa target compound) wavelength and “reference” wavelengths may be verylarge compared to the incremental absorption from compounds of interest.

One method used for gaseous spectroscopy is the use of tunable lasersthat scan through an absorption peak in a short time. This is the coreconcept behind tunable diode laser absorption spectroscopy (TDLAS) thatis already used in commercial instruments. In gaseous samples,absorption peaks are typically very narrow (<<1 cm⁻¹) and high. Thismeans a very narrow tuning range may be used (often <1 cm⁻¹ inwavenumber terms) to cover reference and peak wavelengths. This tuningmay be performed quickly, and with minimum variation in laserconditions.

In liquid systems, on the other hand, absorption bands become farbroader, with lower peak absorptions. This requires tunable systems tocover a far broader range (>10 cm−1, for example)—this is difficult todo consistently. For example, mode transitions within the laser mayoccur inconsistently, leading to sharp changes in power and other beamcharacteristics at the wavelengths of interest.

Similarly, multiple discrete sources operating at wavelengths over therequired range may individually vary in their emission characteristicsover time and operating conditions, leading to apparent changes in“reference” and “peak” transmission and errors in reported chemicalconcentrations.

Furthermore, although it is possible to integrate reference powerdetectors that monitor laser power prior to the sample, such referenceschemes require beam splitting optics which will introduce new opticalartifacts such as fringing into the system; moreover, the power splitoff by these optics may be different from the power delivered to thesample as a result. In addition, such a reference channel will notaccount for optical effects within the sample and sample chamber—whichcan be particularly strong in a coherent, laser-based system.

BRIEF SUMMARY OF THE INVENTION

The present invention describes a system by which infrared lasers,including QCLs, may be used to measure liquid samples, and providessignificant advantages in terms of signal-to-noise ratio in measuringchemical composition of these liquids; as well as resulting in a systemthat is very stable in the face of laser or other optical train changes.

The system includes a liquid handling system that combines reference andsample liquids into a laminar flow that travels through a microfluidicchannel (or “cuvette”) that is infrared-transparent. The system furthercomprises optics to deliver light from one or more infrared lasers intothis channel; infrared light is partially absorbed by the liquids in thechannel according to its chemical constituents, path length, temperatureand optical characteristics. The system further comprises a scanningsubsystem which scans the beam relative to the laminar flow within thechannel, such that the beam scans over both reference and sample fluids.The system further comprises one or more detectors that measure thelight that has been partially absorbed by the liquid in the channel,while the scanning subsystem scans the light over both sample andreference liquids.

Importantly, a microfluidic channel with laminar flow allows liquids tobe presented in nearly identical configurations to the light source, inclose proximity to one another, such that measurements of sample andreference fluids can be made within a short period of time during whichthe system remains stable. In addition, the close proximity of thefluids to one another in a common flow ensures they are presented innearly identical conditions (pressure, temperature, flow rate, etc.).

Detection

In some embodiments, AC-coupled detectors may be used to measure thedifferential absorption between reference and sample liquids at one ormore wavelengths as the scanning subsystem scans the beam between theliquid streams. The scan rate may be adjusted to optimize detector andsystem signal-to-noise ratio (SNR), for example placing it above most1/f noise, but still within the high-response range for the detector andits amplifying circuitry. Various well-known schemes for extracting andfiltering and signal at a specific frequency may be used to optimizeSNR. Inherently change-sensitive (“AC”) detectors such as pyroelectricdetectors may be used; as may other thermal detectors such asthermopiles, or photovoltaic detectors such as cooled or uncooled InGaAsor HgCdTe detectors. Pyroelectric detectors may provide an advantage ofvery high saturation flux (power per unit area) while remainingsensitive to small changes in infrared light as a result of differentialabsorption between reference and sample fluids.

Scanning

Scanning may be achieved by scanning one or more beams optically overthe sample, or translating the sample relative to the beam(s). Manysubsystems for scanning beams over samples have been produced formicroscopy, and similar subsystems may be utilized in the presentinvention.

Lasers

One or more infrared lasers may be used in the present invention togenerate one or more wavelengths of interest. In some cases, a singlefixed-wavelength laser could be used to interrogate a specificabsorption peak of a compound that is not present in the referenceliquid, but potentially present in the sample liquid. As the beam scansbetween reference and sample fluids, the magnitude of the changedetected on the detector allows calculation of the concentration of thecompound in the sample.

In other cases it may be helpful—because of interfering, non-targetcompounds, or because better concentration accuracy is desired—to usemultiple wavelengths, including at least one “signal” wavelength(measuring an absorption peak of interest) and one or more “reference”wavelengths. In such a configuration, these wavelengths may be deliveredsimultaneously from multiple lasers (which may be in a single-chiparray, or in discrete devices), or from one or more lasers that arewavelength-tunable.

When multiple wavelengths are used simultaneously, these may beseparated after transmission through the sample by means of thin filmfilters, diffraction gratings, or similar devices. Alternatively theymay be modulated in such a manner as to make their signals separable inthe detection system.

Relatively broadband laser sources, such as Fabry-Perot lasers, may beused, and component wavelengths split from one another after opticallybefore detection.

The present invention may utilize wavelengths and lasers throughout theinfrared range, including but not limited to the near-infrared andmid-infrared regions where many compounds have characteristic absorptionpeaks, but also in the THz range where stronger laser sources such asQCLs are being developed.

Reference Liquids

The reference liquids used in the present invention may be of severalforms. In the most basic configuration, the reference liquid consists ofa pure sample of the medium contained in the sample liquid—i.e.containing none of the target compound. For example, if the goal is tomeasure impurities (such as hydrocarbons) in water, the reference liquidmay be distilled water, or a known “clean” sample of water from the sitebeing monitored.

In other cases, the reference liquid may contain the compound ofinterest at a desired level; for example in an industrial process wherea compound is added to a liquid medium, a reference liquid mixed toexact concentration in a laboratory may be used. Therefore any signaldetected as the beam in the system is scanned between sample andreference indicates a deviation from the desired level. The phase, orsign, of this signal will indicate whether there is too much or toolittle of the compound, and magnitude will indicate the error level. Aswith many embodiments of the present invention, multiple compounds maybe measured in this manner at multiple wavelengths. For example, anentire “panel” could be run in continuous, real-time fashion in abrewing process—against a “golden sample” of the product.

In another example, a medical liquid such as blood plasma may beanalyzed in the present invention against a standard reference thatcontains target levels of certain constituents, for example glucose. Anydeviations may be measured with high contrast.

In other applications, the reference liquid may be a “before” sample,while the sample liquid is “after,” where chemical change is monitoredover time to measure degradation, for example. For instance, oilcondition in machinery or electrical equipment may be monitored in thismanner to track degradation and call for oil changes or otherpreventative maintenance. Again, the samples are presented in a laminarflow that allows nearly identical measurement conditions, and highcontrast and SNR resulting from the scanning measurement.

In other embodiments, the present invention may be used in aconfiguration where a reference fluid is split into two streams, and onestream is exposed to gaseous, liquid, or solid samples that react withit, alter its chemical composition, or introduce external compounds intoit. The result of this interaction is now the “sample” liquid, which isthen measured as described above. Examples of such interactions includecompounds dissolved from the external sample into the sample liquid,including liquid-liquid extractions, gas-to-liquid extraction,solid-to-liquid extraction. For example, such a system may enablemeasurement of trace amounts of a compound on the surface of a solid, byfirst dissolving this compound in a known liquid, and then measuring theresulting sample liquid against a pure sample of the liquid medium, withhigh contrast as described herein.

In other embodiments, the sample liquid or stream may in fact consist ofthe compounds formed at the interface of two liquids flowing in alaminar system, as a result of reactions between those two liquids. Inthis case, the interface region (“sample”) may be measured at variouslengths into the flow chamber, and the reaction rates/concentrationsdeduced from the rate of growth of the infrared absorption signal fromthe sample stream.

In other embodiments, the reference liquid may be pre-impregnated withcompounds other than those being measured, in order to facilitateaccurate measurement of liquid flow parameters. For example, it may bedesirable to measure the exact cross-section of the sample liquid vs.reference liquid in the laminar flow channel, in order to determinesample concentrations with maximum accuracy. For this purpose, thereference liquid includes a marker that will be missing from the sampleliquid, allowing its omission to be detected in the sample. This markerdoes not necessarily have to function in the infrared—it could, quitesimply, be a color dye that is monitored optically in the visible range(so long as the dye's absorption peaks in the infrared do not interferewith the measurement).

In certain applications, much of the reference liquid may be separatedand re-used at the end of the laminar flow section. Sufficient referenceliquid in the proximity to the sample liquid (enough to account fordiffusion) is stripped and discarded with the sample liquid, and theremainder of the reference liquid is recirculated.

Flow

In many embodiments, a single laminar stream of sample liquid surroundedby reference liquid (either in 2 or 3 dimensions) is required. Such alaminar flow, and the methods and fluidic devices for producing it, arewell known from the fields of microfluidics and cytometry.

In other embodiments, it may be advantageous to produce a multiplicityof laminar sample and reference streams, alternating across the flowchannel. Such a configuration may allow higher SNR in the signalresulting from scanning.

For high transmission in the infrared, it may be desirable to userelatively thin flow channels, for example <1 mm, or in many cases <100microns (um), <50 um, <25 um or even <10 um—depending on thetransmission of the fluid, and the fluid dynamic parameters required tomaintain a laminar flow.

The scanning beam and surface angles of the fluidic chamber may bearranged so as to minimize surface reflections which may interfere withmeasurements by variable constructive or destructive interference andeven potentially feedback to the laser. As most infrared laser sourcesare inherently polarized, the surfaces oriented such that P polarizedlight experiences no reflection as it passes through the measurementchamber.

The present invention may utilize either transmissive or transflective(where light passes through the liquid, reflects, re-passes through theliquid and then back to a detector) configuration.

The present invention may incorporate surface-grazing/evanescentcoupling absorption spectroscopy techniques such as the use of photoniccrystals that are in contact with the sample and reference fluid flows,or more commonly, ATR prisms where the measurement face forms one sideof the fluid flow channel. In such architecture, scanning is stillachieved by moving the beam (which enters the ATR, and reflects at leastonce off the surface in contact with the fluid) perpendicularly relativeto the laminar fluid flow over the measurement face of the ATR crystal.Wavelengths

The present invention may be used throughout the infrared and Terahertzrange where laser sources are available. Specifically, it may be used inthe near-infrared (0.75-1.4 um), short-wave infrared (1.4-3 um),mid-wavelength infrared (3-8 um), long-wavelength infrared (8-15 um),and far-infrared (20-1000 um) regimes where compounds havecharacteristic vibrational absorption lines, and laser as well asdetector components have been developed capable of being used asdescribed above.

QCLs

Quantum cascade lasers (QCLs) may offer specific advantages for use inthe present invention. They may be fabricated to emit at wavelengthsthroughout the mid-infrared as well at the Terahertz ranges where thepresent invention may be used to measure liquid properties. They areavailable in multiple formats, including discrete narrowbandsingle-wavelength devices, broadband (Fabry-Perot) emitters which mayoptionally be combined with wavelength-selective or dispersive elementsto select one or more specific wavelength bands, wavelength-tunablesubsystems, and QCL arrays which may emit a number of wavelengths from asingle-chip device. All of these forms of QCL may be used in the contextof the present invention.

Applications

Applications of the present invention include, but are not limited to:

measurement of medical fluids including blood plasma, urine, or salivaagainst standard reference fluids for diagnostic purposes, or to monitorfor controlled substances; this may include the measurement of bloodglucose level;

measurement of water samples against reference water samples to testfor/determine concentrations of pollutants;

measurement of biological samples against reference media to measurelevels of DNA, RNA, proteins, sugars, lipids, cellular nutrients andmetabolites; this includes measurement of liquids which have surroundedcells or tissue (such as cancer cells, stem cells, embryos) to measureuptake of nutrients and/or production of metabolites; measurement of DNAlevels in polymerase chain reaction (PCR) tests;

measurement of liquid samples from food, drink, or pharmacologicalproduction processes against standard reference liquids to providefeedback for production parameters, measure completion, or measurecontamination;

measurement of liquids used in electrical or mechanical machineryagainst standard reference liquids to measure wear and schedulepreventative maintenance or replacement;

measurement of airborne chemicals through trapping in a liquid stream,and comparison to a pure reference liquid;

measurement of chemical composition in solids through exposure to aliquid, and comparison of that liquid to a pure reference liquid;

measurement of liquids such as milk against a standard reference todetermine nutritional and fat content, and other parameters; measurementof potable liquids such as olive oil against a known reference todetermine authenticity and purity; measurement of potable liquidsagainst reference liquids to measure potentially harmful impurities.

Generalized Liquid Scanning System

More generally, the present invention may be extended to allowmeasurement of liquid-based samples either in flow or non-flowenvironments. The essential elements remain the same: and infrared lasersource such as a QCL (which may operate at one or more wavelengths), amechanism for scanning the beam produced by this source over aliquid-based sample, which may include concentration gradients (thetarget of this measurement system) which result in a variation in theextinction of infrared light as it interacts with the sample (where theaforementioned scanning converts this spatial variation into a temporalvariation in a specific frequency range), a mechanism to guide theresulting infrared light (after scan-modulated interaction with thesample) to a detector subsystem which includes an AC-sensitive detectordesigned to measure changes in infrared light intensity corresponding tothe scanning frequency range; the output of this detector subsystembeing used to calculate concentrations of target substances in theliquid. This scanning may be performed in 1- or 2-dimensions on aliquid-based sample as described below.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other objects, features and advantages will beapparent from the following description of particular embodiments of theinvention, as illustrated in the accompanying drawings in which likereference characters refer to the same parts throughout the differentviews.

FIG. 1A shows the absorption of the target compound;

FIG. 1B shows the absorption of the medium in which the compound isdissolved;

FIG. 1C shows the transmission through the liquid sample;

FIG. 1D shows an idealized case where three narrowband infrared lasersources are used to measure reference and signal absorption frequencies,compute peak absorption, and thereby concentration of the compound;

FIG. 1E shows the reality version of such systems;

FIG. 2 shows presenting the liquid sample in a flow configuration thatallows referencing against a standard.

FIG. 3A shows an example of a scanning pattern;

FIG. 3B schematically shows the transmitted optical power as the beam isscanned over the channel, at three different concentration levels;

FIG. 3C shows the output of an example detector circuit in response tothese optical transmission changes;

FIG. 3D shows the concentration of the target compound calculated in thecurrent system;

FIG. 4 shows a generalized version of the present invention;

FIGS. 5, 6 and 7 show other embodiments of the present invention;

FIG. 8 depicts an example sample holder for use in the presentinvention.

FIG. 9 shows an example of a liquid chamber/channel-integratedattenuated total reflection (ATR) prism that could be integrated into onexample embodiment of the present invention;

FIG. 10A shows an example of a scanned liquid sample in the presentinvention where the liquid includes dispersed solids or liquids;

FIG. 10B shows an example of the present invention used to measuredispersed contents within the liquid sample of FIG. 10 a;

FIGS. 11A-D shows further explanation of the scattering measurement thatmay be employed in certain embodiments of the present invention whereparticles, cells, droplets or other inclusions are dispersed in theliquid sample.

DETAILED DESCRIPTION

FIG. 1 illustrates an example of spectroscopy of a compound in a liquid.FIG. 1a shows the absorption of the target compound, in its pure form,as a function of frequency. In this simplified example, a singleabsorption peak is shown. FIG. 1b shows the absorption of the medium inwhich the compound is dissolved; in this case, a uniform high absorptionis shown (which is the case, for example, for water over certaininfrared ranges). Note the liquid medium may in fact have a very complexabsorption profile with multiple absorption peaks, and may in factconsist of many intermingled chemical components. The present inventionis, in fact, very well suited to handle such scenarios where the mediumhas complex absorption patterns, as it inherently removes commoncomponents between a reference and sample fluid, and therefore thefeatures of the medium in which the target compound is carried (takingthe example where the target is in solution). FIG. 1c then shows thetransmission through the liquid sample, including both the medium andtarget compound. Note the overall transmission may be very low (as isthe case with aqueous solutions in the mid-infrared), and theincremental absorption due to the compound of interest may be extremelysmall. Moreover, with a broadband infrared source such as a glo-bar oreven synchrotron, the power density per frequency is very low, so thetotal power delivered to the frequency range where the compound absorbsis very low. This makes accurate measurement of samples in liquid verychallenging using conventional sources. FIG. 1d shows an idealized casewhere three narrowband infrared laser sources are used to measurereference and signal absorption frequencies, compute peak absorption,and thereby concentration of the compound. FIG. 1e shows the reality ofsuch systems—the laser power may vary significantly over frequency, asmay their bandwidths/band shapes, spatial modes, etc. Thesecharacteristics may also vary significantly with time, temperature,vibration/shock, and other environmental parameters. This means thevariation in laser characteristics overwhelms differential absorptionfrom the compound of interest in many cases, even when great lengths aretaken to stabilize or calibrate the system.

The present invention overcomes this issue by presenting the liquidsample in a flow configuration that allows referencing against astandard, as shown in FIG. 2. A laminar flow is established whichcombines the sample fluid with a reference fluid, and these flow side byside through the optical measurement zone. In the measurement zone, aninfrared beam is translated (scanned) back and forth over the referenceand sample liquids. A laminar flow system, which may be a microfluidicsystem in many applications, ensures that there is not strong mixingbetween the sample and reference liquids; the parameters for such a flow(dimensions, flow rate) are well established in the art. The measurementzone should typically be set in a region where there is a stable slow,but where significant diffusion of the compound(s) of interest betweenthe sample and reference has not occurred (in some cases this may bedesirable, as noted above). The scanning range should be large enough tooptically sample the sample and reference fluids completely, buttypically limited in range in order to maintain substantially identicaloptical path conditions in the system. In some cases the channel itselfmay be translated across the beam, while in others the beam will bescanned over the channel. In some cases, a fluidic chamber may bepre-charged with a laminar flow, the flow terminated, and then thechamber measured optically before significant diffusion occurs. Thechamber itself may be part of a disposable unit, built using low-costmicrofluidic manufacturing techniques. This unit may include thereference liquid on-board, as well as in some cases any liquid requiredto prepare the sample fluid. Note that while the flow cell shown in FIG.2 has two reference flows on either side of a sample flow (which isoften helpful for “centering” the flow), other configurations arepossible. A minimal configuration would merge one sample liquid flowwith a single reference liquid flow (2 inputs), and scanning would occurat the interface of these. More complex flows could include multiplereference and sample flows interleaved.

A brief additional description of the example fluidic measurement unitshown in FIG. 2 is as follows: a sample fluid 201 runs into the chambertogether with one or more reference fluids 202 (one branch is marked)into a chamber with laminar flow 203. In the optical measurement region204 a beam is scanned across the reference fluid as well as samplefluid, with at least one region 205 where it substantially measuresabsorption of the sample fluid, and one region 206 where itsubstantially measures absorption in the reference fluid.

FIG. 3 schematically represents the operation of the system as it isused to determine concentration of a target compound in the samplefluid, in this case using a single infrared laser source and singledetector. FIG. 3a shows an example of a scanning pattern (triangular inthis case, though many other known optical scanning patterns may beused, including 2-dimensional scan patterns) where the infrared beam isscanned from reference fluid, through sample fluid, and back toreference fluid. Note that the beam does not necessarily need to passthrough the entire sample stream; it could simply oscillate on one edgeof the flow between sample and reference fluids. A feedback loop may beused to continuously center the scan optimally on the edge or center ofthe sample flow—this feedback may use the absorption of the compound ofinterest, or other unrelated absorption peaks that are always present(including reference compounds added to the reference or sample liquid,as described above). FIG. 3b schematically shows the transmitted opticalpower as the beam is scanned over the channel, at three differentconcentration levels. Note the incremental absorption as the beam passesover the sample may be extremely small. Note in some cases, the presentinvention may in fact be used to measure the absence or reduction of theabsorption peak in the sample fluid. FIG. 3c shows the output of anexample detector circuit in response to these optical transmissionchanges. The detector and/or circuit are configured in this case to usean AC detection mode, where only changes in optical power are registered(as the derivative of that power with time). Such a configuration mayprovide significant advantages where the incremental absorption is verysmall—it effectively removes the high baseline, and any commonabsorption features. Note that in some cases where the absorption of thetarget compound is high, a conventional DC detection scheme may be used.Even when an AC detection scheme is used, it may be useful to measure DCpower, either with the same detector (through a split AD/DC circuit) orwith a separate detector, so as to normalize the AC signal by the DCoptical power (which will take into account laser power and overallliquid and system transmission, among other long-term changes).Inherently AC detectors such as pyroelectric detectors, which arelow-cost and are stable over temperature, may be used. In fact, a wholeclass of well-known detectors and circuits that have been developed forFTIR instruments (which measure AC signals resulting from a scanninginterferometer) may be employed in the present invention. FIG. 3d showsthe concentration of the target compound calculated in the currentsystem. This concentration could be calculated from a single scan, orfrom the aggregate of many scans, depending on the accuracy andreal-time characteristics required for the application.

FIG. 4 shows a generalized version of the present invention. Amid-infrared laser source 401 produces mid-infrared light 402 that isscanned relative to the sample chamber 405 by a scanning system 403.This scanning system may in fact be a system that translates the samplechamber in relation to a stationary beam. Here the scanning system isshown to scan the beam over a range of positions 404 that pass throughthe chamber windows 406 and the contained liquid sample 407. As the beamis scanned through different portions of the liquid sample, which maycontain concentration gradients of target analytes, the amount of mid-IRlight transmitted at specific wavelengths may vary by transmitted beamposition 408. A de-scanning mechanism 409 serves to deliver all of thislight substantially to the same detector subsystem 411. The de-scanningmechanism may be one and the same as the scanning mechanism, in the casewhere the sample chamber is translated to achieve the scanning, or insome cases a lens with appropriate characteristics may be used to focussubstantially all the scanned light onto the detector element. Thede-scanned light 410 reaching the detector subsystem 411 therefore ismodulated by scanning it through the liquid sample 407, with all otherconditions held substantially identical through the course of the scan.The detector subsystem 411 is an AC-coupled detector system that eitheruses a detector such as a pyroelectric detector which is responsive onlyto changes in optical power, and/or employs a circuit to remove any DCcomponent of the mid-infrared signal from de-scanned light 410 reachingthe detector subsystem. Therefore gain can be applied in order toamplify effects from small changes in transmission due to scannedconcentration gradients, without saturating the output of the detectorsubsystem. The output 412 of the detector subsystem is then processed bya computing unit 413 that calculates absorption and potentiallyconcentrations as a function of position in the sample to generateoutput(s) 414.

The core elements of the invention are: the use of mid-infrared laserssuch as QCLs to produce light at wavelengths corresponding to compoundsof interest in the liquid-based sample; a method of scanning this lightrelative to the sample in order to modulate transmission according tolocal concentrations of these compounds; a method of delivering thetransmitted light to an AC-coupled detector system which amplifies thesetransmission differentials that result from scanning; and a system tocompute absorption and potentially relative concentrations within thesample.

Detectors: examples of detectors include mercury-cadmium-telluride (MCT)photoconductive or photovoltaic detectors run in AC-coupledamplification circuits, or pyroelectric detectors—which are inherentlyAC-coupled in nature. For many applications, pyroelectric detectors maybe well suited because of their AC-coupled nature, very high saturationpower, temperature insensitivity, and low cost. Importantly,pyroelectric detectors remain linear over a wide range of powers(whereas MCT detectors saturate). In particular in a case wheremid-infrared lasers are used, there is often plenty of power, and thepresent invention allows concentration measurements through thedetection of small changes in this power (rather than absolute DC powermeasurement).

Note that the detector subsystem, in addition to the AC-coupled primarydetector(s), may additionally comprise a DC level detector that monitorsthe overall transmitted mid-IR light, and is used to normalize the ACsignal. Such DC-level detection allows adjustment for overall laserpower, system transmission, liquid sample thickness, etc.

Sampling: while many embodiments will use a transmission-type designwhere the scanned beam (where “scanned” is understood to mean either thebeam scanning over the sample, or the sample being scanned relative tothe beam) is transmitted through the sample chamber and the sample.However, the present invention extends to designs employing“transflection” (where the beam passes through the sample, is reflected,and passes through the sample once more on its path to exit), as well assurface-sampling techniques such as attenuated total reflection (ATR)prism-based designs where the beam reflects off a surface in contactwith the liquid sample and evanescently couples into it, evanescentwaveguide designs, and designs where resonant surface coatings (such asphotonic crystal or metamaterial designs) in contact with the sampleamplify interaction between the mid-infrared light and the sample.

Scanning: the beam scanning frequency and pattern will vary byconfiguration and application. Preferably, the scanning allows thesignal corresponding to the absorption, and therefore the concentrationgradient, to be shifted to a frequency well above low-frequency noisesources (1/f noise, etc.) and variations (temperature fluctuations inthe mechanics or laser, etc) in the system and thereby avoid many of thepitfalls of static (DC) transmission measurements systems. For example,the scanning frequency can be at least approximately 1 Hz, 10 Hz, 100Hz, 1000 Hz, 10000 Hz or higher as the detector subsystem allows. Thescanning frequency should also fall into a range where the detectoremployed has sufficient response. For example, pyroelectric detectorsare thermal detectors, and therefore have a roll-off in signal withfrequency that may be pronounced over 100 Hz. The detector circuitshould also be designed—and potentially optimized—for the scanningfrequency. Well-known “lock-in amplification” techniques may be appliedto isolate the signal resulting from the scanning; the phase of thedetected signal relative to the scanning may be used to further refinethe signal. For example, in cases where a known interface between twofluids (say, side-by-side laminar flows of a sample and reference fluid)is scanned, the change in transmitted intensity at that interface may beisolated from other scanning-related optical artifacts. Alternatively, abaseline may be established by running the scan over a section of sampleknown to have no concentration gradients. Various other digitalfiltering techniques that are well known may be applied after theamplified detector signal is captured in an analog-to-digital converter.

FIG. 5 shows another embodiment of the present invention. A mid-infraredlaser source 501 (which may produce one or more wavelengths in themid-infrared) is focused by a lens 502 through a spatial filter 503which is designed to “clean up” the mid-infrared beam, with thetransmitted light well-suited for focusing into a well-defined spot(despite any variation in the output of the laser, such as differentmodes); the filtered light is re-collimated by lens 504 and then scannedover a range of angles by scanner 505. The scanner may scan in 1 or 2axes. The scanned light is focused by lens 506 onto sample holder 507.The scanned beam 507 c (showing two beam positions within the scan)passes through the sample chamber windows 507 a and the containedliquid-based sample 507 b (which in this example, shows two regions withdiffering concentration of a target compound). The sample holder mayoptionally be mounted on a translation stage 508 with one or moretranslation axes in order to position the sample relative to thescanning beam. For example, a “Z” translation (substantially parallel tothe axis of the beam) may be used to optimally focus the beam on thesample within the sample holder, and thereby get maximum contrast duringthe scan; “X” and or “Y” translation may be used to position the samplesuch that the scanning beam traverses specific features havingconcentration gradients of interest (for example, the boundary betweentwo liquid flows, or the location of a biological cell). A capturinglens 509 re-collimates the transmitted mid-IR light and a de-scanningmirror 510 redirects the mid-IR light such that the light remainsincident on the detector 513 with minimal intensity modulation whenthere is no concentration gradient in the sample. A lens 511 focuses thelight, optionally through a spatial filter 512, onto the AC-coupleddetector 513. The detector signal(s) are relayed to a computing system514 that computes absorption gradients, and potentially concentrationsof analytes, in the sample as output(s) 515. This system may alsocontrol laser operation (power and wavelength, for example), scanningand de-scanning modules, and translation stage(s).

Liquids: the present invention may be used to measure liquid-basedsamples of various types, including liquid flows with concentrationgradients, biological cells in liquid, biological tissues in un-driedstate, dispersions of droplets or solid particles in liquids. Eachsample will ideally have concentrations gradients over the scale scannedby the present invention, so as to induce a change in the amount oflight transmitted, and therefore an AC signal on the detector. Thechange in signal may in fact result from the displacement of the medium(for example, water) by a solute or dispersed material, or scattering asa result of the difference of refractive index between a cell, dropletor solid particle and the surrounding medium.

Scattering Measurement: in some embodiments, the present invention maymeasure or calculate scattering resulting from particles or dropletsdispersed in the liquid sample—again by scanning between regions withmore and fewer of such particles/droplets, or between regions where suchparticles/droplets change in nature. In such embodiments, scatteringwith increase as a function of droplet/particle diameter and refractiveindex, which is a function of its composition and wavelength. Throughthe use of appropriate spatial filters before and after the sample, itis possible to isolate or remove scattered light, and thereby calculatescattering from particles/droplets in the liquid in order to deduceaverage diameter (assuming some chemical composition). With multiplewavelengths around infrared absorption peaks for droplet/particleconstituents, it is additionally possible to estimate both chemicalcomposition as well as droplet size as it results from resonant Miescattering (rapid change in scattering as a result of rapid change inrefractive index around a resonant absorption peak for a particularcompound).

For example, in measurements of hydrocarbons in water, often many of thehydrocarbons are not dissolved in the water, but form droplets dispersedin the water. The present invention may be used to measure a sample ofwater with potential hydrocarbon contamination in a laminar flowside-by-side with a pure water reference, by scanning the beam (orequivalently, the sample) back and forth across the interface betweenthese parallel flows. Measurements can be made at several wavelengths,including a peak absorption wavelength for hydrocarbons, but also anon-peak wavelength. Non-peak wavelength signal will indicate scatteringand water displacement; the differential between peak and non-peak willindicate total hydrocarbon concentration. Additionally, if wavelengthson either edge of the absorption peak are measured, the differential inscattering loss (as a result of resonant Mie scattering) may be used tocalculate dispersed hydrocarbon characteristics. Therefore the presentinvention may be used to measure both dissolved and dispersedhydrocarbons in a water sample, and distinguish between these.

FIG. 6 shows another embodiment of the present invention; this exampleshows a system where “de-scanning” onto a single detector element isaccomplished with the use of a short focal length lens 609. A completedescription is as follows: a mid-infrared laser source such as a QCL(which can be a single-wavelength device, emit multiple wavelengths, orhave a tunable wavelength) 601 is collimated through lens 602 (alllenses described in this invention may be refractive or reflective-typelenses), and then scanned using scanner 603 over a range of angles,before being focused on the liquid sample chamber 606 by lens 604. Thesampling spot therefore is scanned over a section of the liquid sampleas indicated by 605; upon transmission through the liquid sample it maybe differentially attenuated depending on chemical concentrations withinthe sample and the interrogating wavelength(s); the beam scanningconverts such gradients into a periodic power fluctuation in thetransmitted light. A collimating lens 607 re-collimates the light, andin this example a fixed folding mirror 608 redirects the collimated beamto a short focal length lens 609. The function of the short focal lengthlens is to focus the transmitted infrared light onto the detector 610.Generally a small detector area is desired, as noise grows with thesquare root of area. In this example, a short focal length is used atthe detector compared to the focal length of focusing lenses 604,607. Asa result, the motion of the beam spot on the detector will be a fractionof the motion of the spot on the sample, allowing a reasonable scandistance on the sample while maintaining focus on the surface of a smalldetector. The signal from the detector subsystem is used by a computerunit 611 that calculates absorption and possibly concentrations, whichgo to output 612.

Detector: In some cases it may be desirable to use detectors withasymmetric dimensions (for example, an elongated rectangle), and toorient this detector with its long axis along the scan direction, tofacilitate complete (or at least consistent) beam capture throughout thescan cycle. In some cases detector arrays may be used in the presentinvention; however, the scanning should not result in beam spot(s)moving from detector element to detector element (which would cause verylarge signal swings not related to concentration gradients in thesample).

Beam Arrays: in some embodiments, multiple beam spots may be used andscanned simultaneously across the sample. These may be multiple spots ofidentical wavelength, split in order to take advantage of increasedperformance from the use of an array of detectors (where the light fromeach beam remains focused on its corresponding detector elementthroughout the modulating scanning described herein). Alternatively, ifan infrared laser array such as the distributed feedback (DFB) QCLdescribed by Capasso et al (DFB Quantum Cascade Laser Arrays, BenjaminG. Lee et al., IEEE Journal of Quantum Electronics, vol. 45, no. 5, May2009) is used, each spot may correspond to a different wavelength ofinterest, and be relayed to its corresponding detector after interactingwith the sample. In one embodiment, QCL DFB array with wavelengthscorresponding to one or more absorption peaks for a target compound,plus one or more reference wavelengths to measure background absorption,can be projected onto a liquid chamber containing a laminar flow withadjacent sample liquid and reference liquids. The laser array isoriented such that the spots from the array run parallel to the flow ofthe liquid, and then the modulating scanning described herein scansthese spots perpendicular to the fluid flow, and across anyconcentration gradient formed by the interface between the sample andreference fluids. After interacting with the fluid and being absorbedaccording to wavelength and concentration, each of these spots isrelayed to a corresponding AC-coupled infrared detector (in many casespart of an array, such as a pyroelectric detector array). The modulationof each detector signal resulting from the modulating scanningcorresponds to the differential absorption between reference and sampleliquid at a particular wavelength; from these signals the concentrationof one or more compounds within the sample liquid may be calculated.

2D Scanning: in the present invention “modulation” scanning (i.e.scanning that is detected by the AC detector module, as distinguishedfor slower stepping/scanning across a sample) may occur in 1 or 2dimensions. A rapid 1-dimensional scan may be used across a particularinterface or feature where there is a concentration gradient. A2-dimensional scan may be used in a pattern to cover an area where thereare concentration gradients. For example, a Lissajous-type scanningpattern may be used to relatively uniformly scan a 2D area of the sample(using simple control electronics). Such a pattern may be used, forexample, where scanning is performed over a single cell in liquid, withvarious wavelengths sampled in order to deduce quantities of one or morecellular components—with the present invention ensuring that even smallquantities of target compounds result in a high-contrast signal at thedetector.

Beam Spot Shaping: various spot sizes and shapes may be used in thepresent invention, including circular spots, but also elliptical spotsparticularly suited for 1-dimensional scanning perpendicular to the longaxis of the elliptical spot. For example, when scanning over theinterface between two liquid flows in a flow chamber, an elliptical spotwith a long axis parallel to the flow (and interface), and thereforeperpendicular to the direction of scanning of the beam over the sample(or sample past beam) may provide particularly high contrast as the spotmoves over the interface between liquids (compared to a more gradualchange for a circular spot, for example). Such a configuration would bevalid for transmission, transflection, or surface-sampling opticalconfigurations such as ATR prisms integrated with the flow chamber.

FIG. 7 shows another embodiment of the present invention; in thisinstance the sample chamber is scanned across the beam in order toinduce modulation according to gradients within the liquid sample. Aninfrared laser source 701 is collimated using lens 702, and focused ontothe sample chamber 705 using lens 704 and mirror 703. The sample isscanned using scanning subsystem 706, which could for example be a piezotransducer (1- or 2-axis) capable of scanning the sample at >1 Hz, >10Hz, >100 Hz or higher frequencies to achieve the signal modulationdescribed herein. A capturing lens 707 re-collimates the beam, which isthen focused onto detector subsystem 710 by focusing lens 709 and mirror708. The signal from the detector subsystem is used by a computer unit711 to calculate absorption and possibly concentrations, which go to thesystem output 712. There are some disadvantages to this embodiment ofthe system, including that the sample holder may have considerable massand therefore require more energy to scan, and scanning may disturb thecontents of the sample holder. However, the advantage is that a veryconsistent optical spot is maintained on the sample, reducing opticalartefacts that result in non-signal modulation at the detector. In thisembodiment, the sample holder may be translated both by the scanningsystem, as well as a secondary translation system that allows the sampleto be put in focus (“Z axis”), and different portions of the sample tobe measured.

Scanning Cytometer: For example, a scanning cell cytometer constructedusing this embodiment may use an XYZ stage to move over a population ofcells within a liquid, and at each cell, the modulating scanningfunction is used to scan a small area containing the cell at highfrequency to cause power modulation on the detector according towavelength and analyte concentrations within the cell. In this manner, alarge population of cells may be measured, each with a very high signalcontrast, allowing high-precision absorption measurements. In this case,the modulation scanning at the cell level may be achieved with either a1-dimensional scan (possible with an elliptical beam with the long axisperpendicular to the scan axis), a 1-dimensional modulating scan withstepping in the direction perpendicular to the scan axis, to build up aprofile of the cell (or integrate the absorption signal), or a2-dimensional modulating scan which uses a pattern such as Lissajousscanning over the local region around the cell—where integration of theresulting modulating signal used to calculate absorption in the cell. Insuch case, an infrared laser source such as a tunable QCL could be usedto sequentially scan the cell at multiple wavelengths in order tocalculate contents of the cell. Alternatively, such a scanning cytometermay be built using an XYZ translation stage for rough samplepositioning, and mirror-based beam scanning mechanisms (as describedabove) to achieve local 1- or 2-dimensional modulating scans over theneighborhood of single cells.

FIG. 8 depicts an example sample holder for use in the presentinvention. As light from laser sources in the mid-infrared is coherentand often has narrow bandwidth (monochromatic), issues of opticalinterference can become problems. In the present invention, where one ormore beams is scanned relative to the sample and sample holder, smallchanges in reflection from the interfaces of the sample holder,compounded by coherent light effects, could cause changes in intensityof the light to the detector that are not related to the sample itself;in addition, strong interference effects within the sample holder maychange the effective optical power at the sample itself (standing waveeffects); finally, reflections back to the laser source (opticalfeedback) may cause significant noise in the laser output. As a result,care should be taken to minimize changes in the optical path through thesample holder, and to minimize reflections from surfaces of this holder.The example shown in this figure consists of an infrared flow cell withsurface angle at the Brewster angle, or the angle where p-polarizedlight is transmitted without reflection through surfaces. Mid-infraredlight 801 (shown here to be p-polarized) from some mid-infrared lasersources (this is particularly true of QCLs) is highly polarized, andtherefore this design may be employed without significant losses orback-reflections. The example sample holder shown here consists of twoinfrared-transparent windows 802 which appear on either side of a liquidsample channel 804, which may contain a stationary or flowing liquidsample. The thickness of the windows 802 is for illustrative purposesonly; typically the thickness of the windows will be many times thethickness of the liquid chamber or channel. The angle of incidence 803from the surrounding medium (typically air) into sample holder windowsurface is at the Brewster angle, where there is no reflection ofp-polarized light; subsequent angles 805 (window-to-liquid) and 806(window-to-air) as well as the angle exiting the liquid into the windoware all constructed, based on the respective refractive indices (at theoperating wavelength) of the surrounding medium, window material, andliquid sample. In this manner the transmitted light 807 is free of“ghost images” resulting from internal reflections, as well as free of“fringes” resulting from resonant cavities inside the sample holder, orbetween the sample holder and other system components. This is ofparticular importance in the present invention due to the modulationscanning of the beam over the sample, and therefore the sample holder.Such scanning may result in slight deviations of incident angle, as wellas scanning over slight thickness variations within the sample holderwindows, and other path length variations, that would be significantlyamplified if resonant cavities were to form inside the sample holder, orbetween the sample holder and other system components. In the presentexample, the beam would be scanned in and out of the plane of the paperrelative to the sample holder (or, equivalently, the sample holder isscanned), so as to keep the incident angles substantially identicalthroughout the scan range.

High-Frequency Laser Modulation. For semiconductor infrared lasersources such as QCLs, spectral inherent linewidths, or width ofindividual lasing modes emitted from the laser, can be extremely narrow(<0.01 cm−1). As a result of these narrow linewidths, resonant effectssuch as fringes may be very pronounced. For semiconductor-based lasersources in the infrared such as QCLs, it is often possible to “spread”the effective linewidth of the laser through the use of some currentmodulation, which produces a rapid thermal modulation within the laserchip, and therefore refractive index changes that result in wavelengthmodulation (and concomitant amplitude modulation). In an extreme case,these lasers may be operated in pulsed mode, where their spectrallinewidth may spread considerably. This is important because a broaderlinewidth reduces the coherence length of the emitted light—or thedistance over which pronounced interference effects may occur. Intraditional infrared spectroscopy applications where gas is measured,narrow linewidth is prized in order to make precise measurements basedon extremely narrow gas absorption lines; however in liquid-phasesamples, absorption peaks typically have peak widths on the order of 5cm−1 or more. As a result, embodiments of the present invention mayinclude modulation or pulsing of the laser light sources in order toreduce coherent artifacts within the system. The modulation of the lasersource should be done at a higher frequency than the modulating scanningdescribed herein, and in fact, beyond the bandwidth of the primarydetector used in the system. Significant thermal tuning (and thereforefrequency broadening) can be achieved in QCL chips, for example, withmodulating frequencies of 10-100 KHz, and even 100-1000 KHz.Additionally, some QCL chips may be pulsed at high frequency, forexample 10-100 KHz and even higher. At these frequencies, thermaldetectors such as pyroelectric detectors do not experience a modulatedsignal, but a DC average of this modulated or pulsed power; thereforenone of the dynamic range of the detector or associated circuitry isconsumed by the modulation or pulsing.

In the context of the present invention, it is desirable to extenddistances between components where back-reflections cannot be avoided todistances beyond the coherence length of the laser source(s).

FIG. 9 shows an example of a liquid chamber/channel-integratedattenuated total reflection (ATR) prism that could be integrated into onexample embodiment of the present invention. Such a configuration may beused in applications where the liquid medium is highly absorptive (suchas water, in large ranges of the mid-infrared range), but narrow liquidchannels that would allow sufficient light transmission are not feasible(because of the danger of clogging, for example). Here a liquid channel901 carrying a flow of liquid is shown; this channel is containedbetween two surfaces: top surface 902 which need not be transparent inthe mid-infrared; and bottom surface 903 which is constructed from aninfrared-transmissive material, and has an integrated ATR prism 904.Incoming infrared light 905 enters the prism (the light and entrysurface may be oriented such that the entry is at the Brewster angle, asdescribed above), and then reflects one or more times from the surfacein contact with the fluid sample. With each total internal reflectionfrom this surface, there is some evanescent penetration 906 of the lightinto the channel and therefore the sample, and absorption according tothe wavelength, the chemical contents of the sample and their resonantinfrared peaks. The transmitted light 907 is then relayed to the ACdetection subsystem as described above. In this design, the beam andsample holder are scanned relative to one another in a directionperpendicular to the plane of the paper, such that the entry angle,reflection angles, and exit angles, as well as the internal distanceswithin the prism, remain identical. A front view depicts a cross-sectionthis from the direction in which the fluid flows, with two beams 908showing the extremes of the scan range, and the liquid showing aconcentration gradient within the range of this scan that will result ina modulation signal at the detector, depending on the incident laserwavelength. This configuration may be used, for example, where a sampleliquid is flowed in parallel with a reference liquid, and the scanningbeam is scanned back and forth across the interface between theseliquids. Any intensity modulation in the transmitted light 907, then,indicates a differential in the contents between sample and referenceliquids—providing high detection sensitivity at a frequency abovelow-frequency noise and system drifts. The example here, again, may beused where a transmission or transflection measurement is notappropriate, because it is physically difficult to flow the sampleliquid through a narrow enough channel (due to viscosity, particulatesthat could cause clogs, etc.).

FIG. 10a shows an example of a scanned liquid sample in the presentinvention where the liquid includes dispersed solids or liquids—forexample hydrocarbons dispersed in a water sample, or fat droplets inmilk. Two incoming infrared beam positions 1001 a and 1001 b (theextremes of a scan range) of a scanned beam 1001 are shown as they aretransmitted through a liquid sample 1003 in a channel or chamber betweentwo infrared-transmissive windows 1002. In this case, the liquid isshown to have two regions that the beam scan range straddles: onewithout, and one with scattering particles such as suspended solids,suspended droplets, biological cells, or other significant inclusionsother than dissolved chemicals. The gradient in such inclusions could bea result, for example, of two liquids in a laminar flow (in or out ofthe plane of the page)—one of which is a sample (typically the one withthe inclusions), and one of which is a reference liquid withoutinclusions, or with a known distribution of included particles ordroplets. As the beam passes through regions with these inclusions,light 1004, 1005 is scattered as a function of the size and shape of theinclusions, as well as the complex refractive index of the inclusionsrelative to the liquid medium carrying them. As described above,specific infrared wavelengths may be used where particular chemicalcomponents of the inclusions (or the medium) have sharp rises or dropsof refractive index (resonant regions), or high absorption. Thus, theability to measure scattered light as a function of wavelength can allowcalculation of various combinations of inclusion size, concentration,and chemical composition—or, be used to calculate the concentration ofinclusions with a particular chemical makeup (for example, resonant Miescattering effects at specific wavelengths could be used to measure onlythe concentration of droplets composed of hydrocarbons, vs gas bubblesor other inclusions in a liquid).

FIG. 10b shows an example of the present invention used to measuredispersed contents within the liquid sample of FIG. 10a . Light from aninfrared laser source 1007 (which, as in all examples in this invention,may provide multiple wavelengths, either sequentially or simultaneously)is collimated by lens 1008 to provide the scanned beam 1001 to theliquid sample 1009. In this example, scanning modulation is achieved byscanning the sample holder with the liquid sample back and forth acrossthe beam. Some light is transmitted directly 1006, with absorptionaccording to concentrations of species in the liquid sample andwavelength. Light that is scattered 1005 due to inclusions in the liquidemerges with a distribution of angles dependent upon the size andchemical composition of the inclusion. In this example, a focusing lens1010 is used to focus the directly-transmitted light through a spatialfilter 1011, such as a pinhole aperture; this aperture transmits lightthat passed directly through the sample with only attenuation, butpreferentially blocks light that has been scattered at an angle byinclusions in the sample. The light transmitted through the pinholeaperture is then detected by a detector subsystem 1012 which isAC-coupled and designed to respond to signals at the frequency of themodulation scanning (of the sample holder past the beam). By measuringthis signal as a function of wavelength, it is then possible tocalculate one or more of total concentration of an analyte in theliquid, concentration of inclusions, contents of these inclusions,and/or size of the inclusions in the liquid sample.

In such scattering-measurement embodiments of the present invention, itmay be desirable to directly measure scattering; for example, invertingthe spatial filter 1011 to block any directly-transmitted light andmeasure only light scattered by the sample as it is scanned across thebeam. This may be repeated at several wavelengths in order to calculateon or more of total concentration of an analyte in the liquid,concentration of inclusions, contents of these inclusions, and/or sizeof the inclusions in the liquid sample. In other embodiments, largelydirectly-transmitted light may be separated from largely scattered lightby use of mirrors and/or spatial filters and measured independently andsimultaneously.

FIGS. 11A-D shows further explanation of the scattering measurement thatmay be employed in certain embodiments of the present invention whereparticles, cells, droplets or other inclusions are dispersed in theliquid sample. For each graph, the horizontal axis is optical frequency,with higher frequencies (shorter wavelengths) to the right of thegraphs. Graph 1101 shows the absorbance, as a function of wavenumber, ofan example compound, with a resonant absorption peak centered at ν_(a).For standard absorption measurements in the present invention, a lasersource would be configured to emit infrared light corresponding to thispeak, and the beam scanned over the sample containing potentialgradients in concentration of this peak, resulting in a modulation ofthe transmitted light (as a result of the compound-specific absorption).Light one or more other wavelengths, typically nearby to the targetabsorption peak, may also be used to establish a “baseline” for the peakabsorption measurement (i.e. cancel out other factors and overlappingabsorption signatures—not shown here).

Graph 1102 shows the real refractive index of the target compound (solidline) and liquid medium (dashed line) as a function of frequency. As aresult of the Kramers-Kronig relationship between real and complexrefractive index, the real index of the target compound displays a“wiggle” that is a derivative of the absorbance shown above it, inaddition to a constant term. In this illustration, the index of themedium is relatively constant. As a result there is relatively rapidchange (with frequency) of index differential between the targetcompound and the medium, with a local maximum at ν_(b) and a localminimum at ν_(c). The importance of this variation in indexdifferentials becomes clear in graph 1103, which represents thescattering efficiency of a droplet or particle of the target compoundresident in the medium. The scattering is a function of the size of theinclusion (held constant for the purpose of this illustration) vs theilluminating wavelength, as well as the refractive index differential.There is a general upward trend towards higher frequencies (shorterwavelengths), as the size of the particle becomes larger compared to thewavelength. Superimposed on this scattering “baseline” is the localvariation due to the refractive index change around the resonantfrequency of the compound (really specific molecular bond vibrationmodes within the compound). Where index differential is higher (ν_(b)),scattering increases, and where it is lower (ν_(c)), scatteringdecreases. This effect—resonant Mie scattering—occurs over a shortfrequency range where other factors are relatively constant. As aresult, in certain embodiments of the present invention, as describedabove, it is possible to measure compound-specific scattering in aliquid sample. As described above, substantially directly transmittedand scattered light may be measured separately, or the combined effectsmay be measured, resulting in an extinction curve such as the one shownin graph 1104. In this compound signal, one or more discrete frequencypoints may be used to measure the characteristics of the liquid withdissolved or dispersed components: frequencies ν₁ and ν₂ may be used tomeasure non-specific scattering from the sample (and therefore indicate,generally, the level of inclusions in the liquid); a laser at frequencyν_(a) may be used to assess absorption (at this frequency there is nonet effect from resonant Mie scattering, but includes the baseline Miescatter) alone when baselined using non-resonant scattering measurementsfrom ν₁ and ν₂. Finally measurements at frequencies ν_(a) and ν_(b) maybe used to extract the resonant Mie scattering effect, and thereforecompound-specific scattering by inclusions in the system. Thesemeasurements, made using the scanning modulation system described in thepresent invention may enable high accuracy calculation of dissolved anddispersed components within a liquid sample.

Wavelengths: the present invention is primarily focused on themid-infrared (2-20 um) wavelength range where molecules have specificresonant absorption fingerprints; furthermore the invention may beapplied in the Terahertz range (100-1000 um) to which infrared lasersources have recently been extended, and where molecules likewiseexhibit characteristic fingerprints. In this range, it is also possibleto measure interactions between molecules, or within molecules (such asproteins, when folding) using the spectroscopic techniques describedherein. The present invention may be used, for example, to scan theinterface between two liquid samples that interact, providing highsensitivity to the resulting molecular interactions provided by thescanning-modulated liquid measurement system described herein.

Laser Sources: the present invention comprises infrared and Terahertzlaser sources of all types—the key distinguishing features of suchsources (as opposed to traditional incandescent or even synchrotronsources) being that: they provide relatively high power at specificwavelengths of interest; and they are coherent, small aperture sourcesthat are a result may be efficiently collimated or focused onto asample, and therefore provide relatively high optical power onto alimited area, which is then scanned to provide the modulation that iscentral to the invention. Specifically, quantum cascade lasers (QCLs)are a suitable source for many embodiments of the present invention, asthey can be manufactured to emit light at tailored wavelengths withinthe mid-infrared and Terahertz bands that are the subject of the presentinvention. Furthermore, QCL sources may be tunable (through the use ofexternal gratings, tunable filters, or other mechanisms) over wavelengthranges suitable for measuring resonant absorptions in liquid-phasesamples; furthermore, monolithically integrated arrays of QCLs withdistinct wavelengths may be fabricated, again emitting over a rangesuitable for liquid-phase sample measurement. All of these types may beused in the present invention. Other infrared laser sources, includingCO₂ lasers, lead-salt lasers, optical parametric oscillators, etc. maybe used in the present invention.

Example Embodiment—Hydrocarbon in Water Measurement:

The present invention may be used to measure impurities in liquids, forexample hydrocarbons that may be present in water as a result ofhydrocarbon exploration, exploitation or processing operations. Anexample embodiment for this application comprises the following:

A mid-infrared QCL source configured to emit at a frequency around amajor hydrocarbon absorption band, for example 1460 cm−1. This QCLsource is tunable such that it covers a range that includes thehydrocarbon absorption band, but also adjacent frequencies wherehydrocarbons do not absorb as strongly (for reference levels). This QCLsource may be pulsed, or modulated at high frequency (for example, 100kHz) to spread its bandwidth and avoid some coherent artifacts in thesystem.

A liquid handling system that introduces the liquid sample, along with areference liquid (pure water) into a flow chamber where these liquidsflow in laminar fashion through a measurement cavity.

One surface of this flow chamber is bordered by an infrared-transparentwindow, for example CaF2 or ZnSe. This window has integrated into it aATR prism which allows multiple internal reflections of infrared lightfrom the surface in contact with the fluid chamber, these reflectionsoccurring along the axis of the flow.

Optical components to relay the infrared light from the QCL source intothe ATR prism, with the center position of the reflections in the ATRbeing close to the interface of the sample and reference liquid flows;the entry angle into the ATR and the exit angle out of the ATRconfigured relative to the polarization of the QCL source such that aminimum reflection occurs at these surfaces, according to the Brewsterangle calculated using the index of the ATR prism material;

A sample scanning system that repetitively translates the sample holderand ATR in a direction perpendicular to the flow and to the sequence ofreflections inside the ATR. This scanning system translates the sampleand the contained flow at roughly 100 Hz, for example.

Optics to capture relay the light emerging from the ATR prism, which hasevanescently interacted with the flow in the chamber, to a detectorsubsystem;

A detector subsystem which is configured to detect the transmittedinfrared light, with electronics designed to isolate and amplify thesignal that results from the scanning of the sample holder (andcontained flow) and therefore the effect of the hydrocarbonconcentration gradient at the border between the sample and referenceliquid flows; further comprising a DC level detector which measures theaverage power transmitted through the system; for example, the ACdetector in this system may be based on a pyroelectric detector (whichis inherently AC-sensitive); the DC portion may be based on a thermopiledetector; both of these are uncooled, stable, broadband and low-costdetectors;

A control and computing system which:

-   -   tunes or switches the QCL source sequentially to wavelengths        corresponding to one or more reference wavelengths (where        hydrocarbon in relatively weak) and peak absorption wavelength        (where hydrocarbons in question have relatively strong        absorption);    -   optionally controls the modulation scanning of the beam between        the sample and reference flows, within the ATR sample;    -   records the amplitude of modulation detected by the detector        subsystem, as well as the DC power level transmitted through the        system; normalizes the modulated power by the DC transmission;    -   calculates hydrocarbon concentration in the water by normalizing        signal at peak absorption wavelength by the signal at reference        wavelengths;    -   reports the hydrocarbon concentration in the sample;    -   optionally, controls the scanning or other translation mechanism        to position the flow interface (between sample and reference) at        the centerpoint of the beam vs sample holder scanning range;    -   optionally, occasionally positions the scanning range entirely        in the reference liquid, so as to extract a baseline signal        level where no concentration gradients are present;    -   optionally, stops all scanning motion and centers the beam on        the flow interface to observe signal from any turbulence within        the flow, adjusting flow rates appropriately to achieve laminar        flow and therefore a clean interface between the two fluid        streams.

This example system enables the measurement of very low levels ofhydrocarbons dissolved in water samples through the use of the presentinvention's unique infrared laser liquid-scanning plus AC detectionarchitecture.

Example Embodiment—Scanning Cytometer:

The present invention may be configured to measure biological cells in aliquid sample between two slides—a standard format for biologicalresearch and clinical diagnostics such as inspection of cells from a papsmear for cancer. In this example, the slides are transparent to bothvisible and infrared light; for example they may be CaF2 windows. Thisexample system comprises:

a visible microscopy system which images the cell or tissue sample,which may have dyes or fluorescent labels applied to it for the purposeof increasing contrast and/or identifying certain cell characteristics;

a translation system which allows imaging or infrared measurements ofdifferent regions, laterally, across the cell sample, and focus of thesample for both visible imaging and infrared measurement;

a system which calculates, based on position in the visible image(s),the position of a cell with respect to a scanning infrared cytometrysubsystem, so as to bring any cell identified in the visible image intofocus for infrared scanning as is described in the present invention;

a QCL source capable of emitting multiple mid-infrared wavelengthscorresponding to absorption bands for molecules found in cells, such as(but not limited to) DNA, RNA, lipids, proteins, cell metabolites, cellnutrients; as well as nearby bands that serve as reference wavelengthsfor baseline measurements;

optics configured to focus the QCL output through a pinhole aperture forthe purpose of “cleaning up” any higher mode emissions from the QCL(s),in order to ensure a consistent, uniform spot may be focused on thesample;

optics configured to form an elliptical spot on the sample, with a longaxis perpendicular to the modulation scan direction; the elliptical spotdesigned to provide high contrast as the beam is scanned over a cell,but relatively uniform signal for small displacement of the beam vs thecell perpendicular to the scan axis; enabling single-axis modulationscanning of each cell;

optics configured to scan the beam rapidly such that the spot on thesample is sufficient to scan across a single cell, perpendicular to thelong axis of the elliptical spot as described above; this modulationoccurring at a frequency above much of the low-frequency noise andvariations in the system (for example, 100 Hz);

optics configured to capture and “de-scan” the transmitted light so thatangle or position variations induced by the scanning system describedabove are reversed, such that light may be relayed to stationarydetectors;

optics configured to split substantially light that was substantiallydirectly transmitted through the sample (with some attenuation) fromlight that was substantially scattered at an angle as it passed throughthe sample and any cells in the infrared optical beam;

detectors configured to respond preferentially to signals at thescanning frequency, to measure signals associated with changes intransmitted light as the beam passes through a cell, and thereby localvariations in infrared light absorption and/or scattering associatedwith the cell; at least one of these detectors configured to measuresubstantially transmitted light, and another one of these detectorsconfigured to measure substantially scattered light;

a computing system configured to receive the detector outputs andcalculate absorbed and scattered light from the signals;

a computing system which, based on the calculated absorption andscattering signals at different wavelengths, calculates cellular contentof one or more compounds within the cell being measured;

a control system which, based on cells identified in visible images,positions the infrared scanning system at cells, tunes or switcheswavelengths in the QCL source, and scans cells with the infrared beam,performing the measurements described above.

Such example system may be used in an automated diagnostic applicationto further inspect suspicious cells (based on visible images ofdye-marked cell samples) for DNA content and/or distribution, which mayidentify cells as likely cancer cells based on an abnormal amount orspatial distribution of DNA or other materials.

For such cancer screening, additional measurements can be made withembodiments of the present system which measure peak frequency of DNAbond vibrations in order to measure packing density (which affects bondvibration frequencies).

Additionally, absorption/scattering peaks corresponding to specific DNAbases (cytosine, in particular) may be measured in order to estimatemethylation level.

One embodiment of a scanning cytometer system utilizes the scanningsystem as described to inspect individual reproductive cells (gametes)in order to determine the likelihood of reproductive success and/oroffspring viability for human as well as livestock reproductivemedicine. The scanning cytometer is configured with mid-infrared lightsources (such as QCLs) with wavelengths corresponding to criticalmolecular components within the gametes. For example, one or morewavelengths corresponding to DNA molecular vibrations may be utilized inorder to (1) quantify the absolute amount of DNA carried by the cellusing extinction (absorption and/or scattering) at the peak frequency,in order to characterize DNA base count, which may indicateaneuploidies, and may also be used in sperm cells to determine the sexof the prospective offspring using the X-Y chromosome DNA differential;(2) detect the packing density of the DNA by its spectroscopicscattering signature (resulting from differences in overall DNA massshape and size) and/or by shifts in the frequency distribution of itsabsorption and/or scattering peak wavelengths (resulting fromdifferences in stresses on the bonds being observed); (3) the level ofDNA methylation in the cell, as an indicator of potential epigeneticfactors.

These methods, besides being applied pre-fertilization to gametes, maybe applied to Zygotes and embryos in order to profile them and predictreproductive success as well as the success of an offspring.DNA-specific measurements, together with non-DNA measurements tocharacterize other components of the embryo, as well as density, size,shape, may be used to select specific embryos for implantation.

Scanning Flow Cytometry System

Embodiments of the present invention that use a scanning beaminterrogating a flow in order to measure single particles or cells arealso possible. For example, a microfluidic flow cell may be used tocreate a laminar flow containing a core stream surrounded by a sheathstream, with the core stream containing particles or cells. A beam isfocused on the area surrounding this core stream. The beam may be shapedsuch that it is asymmetrical.

In such a configuration the beam will typically be scanned at afrequency that allows multiple passes over each particle as it transitsthe interrogation region, allowing the signals corresponding toabsorption or scattering to be isolated with high signal to noise.Depending on the shape of the beam, additional information about theshape of the particle may be extracted from the scan signal or resultingsignal envelope.

In some embodiments of the present invention, it may be desirable toemploy two or more scanners to direct beams of different wavelengths, attwo or more different frequencies that may be clearly separatedelectronically after detection, using appropriate bandpass filtering.

In other cases, spots of different wavelengths may be scannedsimultaneously, but offset from each other in a manner that allowsseparation in after detection.

Time-Resolved Measurements

The present invention is particularly useful for time-resolvedmeasurements of particles, cells, or groups of cells (includingembryos). The advantage of a scanning cytometry system, of course, isthat repeated measurements of single or multiple cells may be made totrack changes over time, including changes in: chemical composition andconcentrations, molecular conformation/folding/condensation, number ofcells in a particle, volume, shape, density, and orientation.

However one known problem with time-resolved measurements inconventional scanning cytometry is photobleaching and other photodamage.Photobleaching results from repeated exposure to excitation wavelengthsused to excite fluorescent dyes and labels—leading to lower fluorescentresponse and therefore inaccurate measurements. Moreover, the shortwavelengths often used as excitation wavelengths may cause directionization damage to the particle, cell, embryo or organism underobservation. Finally, dye- and label-based measurements suffer from thefact that concentration or effectiveness of these external chemicals mayvary over time and depending on local condition, causing further errorsin measurements.

The present invention enables time-resolved measurements without theseproblems, and therefore without their negative effects on accuracy. Thepresent invention uses mid-infrared radiation, allowing directmeasurements of chemical concentrations and conformation, and associatedmeasurements such as non-water cell volume, shape, etc.—all withoutexternal labels or dyes. Therefore there is no dependence on labels thatmay photobleach or change in effectiveness/concentration over a timeseries measurement. Furthermore, the very low energy of the radiationused in the present invention precludes any ionization, and thereforeremoves any question of cellular damage due to interrogating radiation.

A number of time-resolved measurements may be made with the presentinvention, including but not limited to drug-cell interactions(including drug uptake, drug efflux), metabolic measurements, celldivision, apoptosis, chromatin condensation, cell-cell interactions,embryo growth, lipid and other product generation, internal proteinchanges, changes in local chemical environment as a result of cellactivity, and shape, volume, density, and cell count changes.

Example Embodiment—Drug/Cell Interaction Measurement

The present invention may be used to measure drug interactions withcells, for example the effects of drugs on cancer cells. In such anembodiment, cells would be placed in a plate containing multipleindividual wells into which nutrients as well as drugs are placed. Thesecells are scanned using one or more mid-infrared wavelengthscorresponding to specific compounds within the cells, for examplenucleic acids, proteins, lipids, or metabolic products. Typically, therewill be additional wavelengths that act as “reference” wavelengths—toeliminate cross-effects from other compounds, or to eliminate effectsfrom changes in cell shape or volume. A wide range of wavelengths may beused (employing one or more tunable quantum cascade lasers, for example)to measure the cells, using scanning, over a broad spectrum, and thenknown data techniques such as principal component analysis, or newmethods such as “deep learning” neural networks, may be used to classifychanges in cells. In such a manner, changes in cell populationsresponding to drugs may be classified with high accuracy, and withoutlabels or dyes that could disturb cell behavior, or create their ownsources of error over time, as described above.

In such an embodiment, the scanning system described herein mayadditionally be used to measure the relative concentration inside andoutside of the cell of one or more compounds, for example to measureuptake of a compound, or efflux of a compound into the well containingthe cell. Such compounds could include drugs, nutrients, metabolicproducts, or others. Because of the scanning nature of the presentinvention, the signal (at a particular wavelength) will be proportionalto the relative extinction (absorption and scattering), and thereforecan give relative concentration measurements between the cell(s) andits(their) local environment. For example, at a certain point in themeasurement series, a compound could be added to the cell, and the timerequired for the concentration within the cell(s) to rise could bemeasured using the present invention.

Example Embodiment—Embryo Monitoring and Scoring

In on embodiment of the present invention, the scanning system is usedto score individual embryos for potential implantation in human oranimal reproductive medicine. In in-vitro fertilization (IVF) proceduresit is preferable to implant a minimum number of embryos to avoidmultiple births, and therefore to select the most viable embryos forimplantation. Current selection procedures are very limited because theymust minimize damage to the embryo, and therefore use of dyes or labels(or excitation lasers with high photon energy) is not advisable. As aresult, selection technologies are centered primarily on visible imaging(whether with a human viewer or an automated image processingalgorithm), or measurements of the medium in which the embryos reside tomeasure uptake or nutrients and/or metabolic products. There is noability to directly measure chemical changes within embryos, or achemical-structural changes (for example, nuclear content/activityseparate from other cellular components).

The present invention allows direct measurement of the chemicalcomposition and evolution of the candidate embryo over the first days ofgrowth, and therefore a far larger range of parameters of the embryo maybe observed: chemical composition; structure; and combination ofchemistry and structure through resonant scattering measurements.

Scattering and Resonant Scattering Measurement

In a resonant scattering measurement, infrared wavelengths aroundabsorption peaks of specific molecules are used in scattering/angledeflection measurements; at these wavelengths, the refractive index ofthese molecules varies (in accordance with the Kramers-Kronigrelationship); therefore particles or cells will refract lightdifferently at specific wavelengths in accordance with chemicalcomposition as well as structure. Such chemical-specific (resonant)scattering is known as resonant Mie scattering.

Scattering may be measured in the present invention through the use ofspatial masks in the illuminating beams that block directly-transmittedlight from a detector; they may in addition use spatial masks to limitthe angle distribution of the incoming light to the sample, and thenanother spatial mask to sample specifically those angles where light wasblocked after the sample; any light in the output will then be a resultof scattering from the sample. The scattering signal will, in accordancewith the present invention, be present in the detector at a specificfrequency, and therefore may be isolated with high signal-to-noiseratio. Depending on the wavelengths employed and angles measured, thisconfiguration of the present invention may measure particle/cell shape,volume, density and also chemical-specific volume, density and packinginformation.

Example Embodiment—Sperm Diagnostics and Selection

For human fertility treatments as well as livestock breeding it is oftendesirable to measure sperm cell parameters to perform diagnostics and/orselect individual cells for fertilization. An embodiment of the presentinvention may be used to first map a large number of cells in a samplecontainer (using a scanning beam and/or translation of the container),and then to measure individual cells, for diagnostics as well asselection.

In such a system, the beam may be scanned over the cell in a patternthat allows chemical/structural information (such as DNA amount, forploidy and X/Y information, DNA packing, DAN alpha/beta configuration,DNA methylation, cell volume and shape) to be measured, but also allowsthe system to measure, based on the timing of the resulting signal, therelative position of the cell to the center of the scan pattern, andthereby allows a control system to track the cell with the beam as thecell moves through the sample chamber. This ultimately allows anaccurate measurement of a moving cell, and at the same time allowsmeasurement of the progressive motility of the cell, another importantmarker for sperm cell viability.

Alternative Scanning Mechanisms

The present invention may use conventional electromechanical methods,such as galvanometers, to scan the beam over the sample. It may alsoemploy solid-state methods, such as the use of modulators, including butnot limited to acousto-optic modulators or deflectors that use RFsignals to tune diffractive patterns in a material and thereby deflect abeam passing through it. The present invention may also use fixeddiffraction gratings, which when the incident wavelength is changed,diffract light at different angles. In such a configuration, slightmodulation of the source wavelength (for example, thermal modulation ofa QCL) would cause slight deflections of the beam coming into a sample,and thereby provide the scanning mechanism in the present invention.

Scanning mechanisms may provide either continuous scan patterns (such asa sine wave) or discrete point scanning (as might be the case with anacousto-optic deflector which deflects a beam to discrete angles). Inthe discrete case, typically one position would correspond to the sampleunder measurements, and another position would correspond to thereference medium surrounding the sample.

Beam Shaping

As described, the present invention may employ a range of scanningpatterns, including 1- and 2-dimensional scan patterns. In addition, thepresent invention may employ various shapes of beam cross-section(“spot”) at the sample location. For example, if a 1-dimensional scan isused to scan a particle, a spot with a relatively small diameter alongthe axis of the scan, and relatively long diameter in the perpendicularaxis may be used, in order to get maximum contrast while scanning overthe particle while preserving uniform sampling of the particle in theother direction (making the measurement more position-independent).

While various embodiments have been particularly shown and described, itwill be understood by those skilled in the art that various changes inform and details may be made therein without departing from the scope ofthe invention as defined by the appended claims.

1-8. (canceled)
 9. A method of analyzing an analyte comprising: a)providing the analyte in liquid to an analyzer, the analyzer comprising:at least one optical source comprising a first light; at least oneoptical detector; a first optical path; and a second optical path; b)emitting the first light from the at least one optical source; c)guiding the first light along the first optical path to the analyte inliquid and along the second optical path to a parallel flowing referenceliquid, wherein the parallel flows are at substantially identicalconditions; d) guiding the resulting, transmitted light from the firstoptical path and the second optical path to the at least one opticaldetector; e) measuring the difference between the transmitted light fromthe first optical path and the second optical path; and f) computing acharacteristic of the analyte based on the difference between thetransmitted light from the first optical path and the second opticalpath.
 10. The method of claim 9, wherein the computed characteristic ofthe analyte is selected from the group consisting of foldingconformation, alpha/beta configuration, methylation, and degree ofcondensation.
 11. The method of claim 10, wherein the analyte is aprotein.
 12. The method of claim 10, wherein the analyte is DNA or RNA.13. The method of claim 9, wherein the first and second optical pathsare alternated.
 14. The method of claim 9, wherein step e) comprisesdetecting optical interference between the transmitted light from thefirst optical path and the second optical path.
 15. The method of claim9, wherein the at least one optical source is a quantum cascade laser(QCL).
 16. The method of claim 15, wherein the QCL iswavelength-tunable.
 17. The method of claim 16, wherein the QCL emits atleast two wavelengths such that the first optical path results in afirst and a second transmitted light and the second optical path resultsin a third and a fourth transmitted light.
 18. The method of claim 17,wherein the computed characteristic of the analyte is selected from thegroup consisting of concentration, chemical signature, and structure.19. The method of claim 17, wherein the computed characteristic of theanalyte is selected from the group consisting of folding conformation,alpha/beta configuration, methylation, and degree of condensation. 20.An apparatus for analyzing an analyte, the apparatus comprising: a) atleast one optical source that emits a first light; b) at least oneoptical detector; c) a first optical path; d) a second optical path; e)a flow chamber for receiving the analyte in liquid and a parallelflowing reference liquid, wherein the parallel flows are atsubstantially identical conditions; and f) a computing unit; wherein thefirst light is guided along the first optical path to the analyte inliquid and the along the second optical path to the reference liquid;wherein the at least one optical detector detects and measures thedifference between the resulting, transmitted light from the firstoptical path and the second optical path; and wherein the computing unitcomputes a characteristic of the analyte based on the difference betweenthe transmitted light from the first optical path and the second opticalpath.
 21. The apparatus of claim 20, wherein the computing unit computesthe folding conformation, alpha/beta configuration, methylation, ordegree of condensation of the analyte.
 22. The apparatus of claim 21,wherein the analyte is a protein.
 23. The apparatus of claim 21, whereinthe analyte is DNA or RNA.
 24. The apparatus of claim 20, wherein thefirst and second optical paths are alternated.
 25. The apparatus ofclaim 20, wherein the optical detector detects optical interferencebetween the transmitted light from the first optical path and the secondoptical path.
 26. The apparatus of claim 20, wherein the at least oneoptical source is a quantum cascade laser (QCL), and optically awavelength-tunable QCL.
 27. The apparatus of claim 26, wherein the QCLemits at least two wavelengths such that the first optical path resultsin a first and a second transmitted light and the second optical pathresults in a third and a fourth transmitted light.
 28. The apparatus ofclaim 27, wherein the computing unit computes the concentration,chemical signature, or structure of the analyte, wherein the structureis selected from the group consisting of folding conformation,alpha/beta configuration, methylation, or degree of condensation of theanalyte.